1. Field of the Invention
The present invention relates to a microscope, particularly a sophisticated microscope using lights of two wavelengths including at least infrared ray or near infrared ray where one light corresponds to the resonance wavelength between the ground state and a given vibration state of a molecule or a given crystalline structure in a sample, and the other light corresponds to an exciting wavelength between the given excited state and a higher electron excited state, and then, by overlapping the lights partially in space and irradiating on the sample, an optical response (e.g., fluorescence) from the sample is observed in a minute region smaller than the diffraction limit of the resulting overlapped light, to detect a chemical group of the sample supersensitively which is characterized by a given vibration.
This invention also relates to a microscope using two kinds of light such as a pumping light and an erasing light where in a super resolution state to repress a fluorescent light-emitting region which is obtained by the pumping light under the diffraction limit by the erasing light, a background signal which results from a scattered light or a side-excitation process by irradiating the erasing light for a sample to be observed, and thus, the spatial resolution and thus, the image quality for the sample is developed.
2. Description of the Relates Art
Technology of optical microscopy has long been developed, resulting in the invention of various types of microscopes. Moreover, microscope systems with improved performance have been developed in recent years due to advances in related technologies beginning with laser technology and electron imaging technology.
Recently, a fluorescence microscope having a high spatial resolution beyond a diffraction limit by using double-resonance absorption is proposed in Japanese Patent Application No. Kokai Hei 10-142151 (JP A 10-142151). FIG. 1 is a conceptual view showing the process of double-resonance absorption that occurs in a molecule. The molecule in the ground state S0 is excited to S1, which is a first excited state, due to light at wavelength λ1. Furthermore, this shows excitation to S2, which is a second excited state, due to light at wavelength λ2. This also shows the case of extremely weak fluorescence from S2.
Extremely interesting phenomena occur in the case of a molecule that has the optical properties indicated in FIG. 1. FIG. 2 is a conceptual view of the double-resonance absorption process, in the same manner as FIG. 1, wherein the horizontal X axis indicates width of spatial distance, spatial region Al being irradiated by light of wavelength λ2, spatial region A0 not being irradiated by light of wavelength λ2.
In FIG. 2, numerous molecules are generated in the S1 state due to excitation by light of wavelength λ1 at spatial region A0, and then fluorescence is visible due to light emission at wavelength λ3 from spatial region A0. However, since spatial region A1 is irradiated by light of wavelength λ2, most molecules in the S1 state are immediately exited to the high S2 state such that molecules in the S1 state aren't present. This type of phenomenon is confirmed for any number of molecules. By this means, even if fluorescence of wavelength λ3 entirely disappears, fluorescence itself at the A1 region becomes entirely controllable since there was no fluorescence originally from the S2 state. Therefore fluorescence occurs only in the A0 spatial region.
This result has extremely important meaning when considered from the standpoint of the applied field of microscopy. That is, in a conventional scanning-type laser microscope, etc., a laser light is concentrated into a micro-beam by means of a condensing lens and then, is scanned across a sample to be observed. During this process, micro-beam size becomes that of the diffraction limit determined by wavelength and the numerical aperture of the condensing lens, and spatial resolution better than this limit can't be anticipated.
However, in the case of FIG. 2, two types of light (wavelength λ1 and wavelength λ2) are skillfully combined spatially, and the fluorescence region is controlled by irradiation of light of wavelength λ2. Therefore, for example, upon consideration of the region of irradiation of light of wavelength λ1, the fluorescence region can be made narrower than even that of the diffraction limit determined by wavelength and numerical aperture of the condensing lens. Therefore this principle can be utilized to make possible a super-resolution microscope (e.g. a fluorescence microscope) using double-resonance absorption to exceed the diffraction limit.
Moreover, for developing the super resolution of a super resolution microscope, such a technique is disclosed in Japanese Patent Application No. Kokai Hei 11-95120(JP A 11-95120) as employing a fluorescence labeler molecules or two types of light of wavelength of λ1 and wavelength of λ2, in order to enhance the functions of the super resolution microscope. In this technique, three quantum states including at least a ground state are defined. Then, to a sample to be observed are combined fluorescent probe molecules to dye various molecules which are transited through thermal relaxation rather than fluorescent thermal relaxation in the deexcitation to the ground state from the higher energy level excited state without the first excited state and biomolecules to which a dying technique is performed biochemically. Then, the sample is excited to state S1 by the light of wavelength of λ1 to excite the molecules for dying, and subsequently, excited to a higher energy level quantum state. In this case, the fluorescent light can be repressed. In this way, if the spatial fluorescent region is narrowed artificially, the spatial resolution of the super resolution microscope can be developed.
Such an optical property of molecule as mentioned above can be explained quantum chemically. That is, generally molecules are comprised of various atoms bounded by π or σ bonds. In other words, molecular orbitals of a molecule exist as π molecular orbitals or σ molecular orbitals, and electrons present in these molecular orbitals take on the important role of bonded the various atoms. Among such molecular orbitals, electrons of σ molecular orbitals strongly bond the various atoms and determine inter-atomic distances within the molecule that form the skeleton of the molecule. In contrast, an electron in a π molecular orbital contributes almost nothing to bonding of the various atoms and instead restrains the entire molecule with an extremely weak force.
In many cases, when light excites an electron in a σ molecular orbital, inter-atomic spacing of the molecule changes greatly, and large structural changes occur which include dissociation of the molecule. As a result, kinetic energy of the atoms and energy imparted to the molecule by light are mostly changed into thermal energy due to structural change. Therefore excitation energy isn't consumed in the form of the light called fluorescence. Moreover, even if fluorescence were assumed to occur during this process, the duration of such fluorescence would be extremely short since structural change of a molecule is extremely fast (shorter than the order of picosecond).
In contrast, an electron in a π orbital has the property of excitation with nearly no effect upon molecular structure itself, staying for a long period of time in an excited quantum state of high quantum discrete position, and subsequent decaying by emission of fluorescence on the order of nanosecond.
According to quantum chemistry, possession of a π molecular orbital by a molecule is equivalent to possession of a double bond so that an essential condition for selection of the utilized fluorescent labeler molecule becomes the possession of abundant double bonds. However, even among molecules having double bonds, six member rings such as benzene and pyridine have been confirmed to have extremely weak fluorescence from the excited state S2 (e.g., M. Fuji, et al., Chem. Phys. Lett. 171(1990)341).
Therefore if a molecule having six member rings, such as benzene, pyridine, etc., is selected as a fluorescence labeler molecule, the lifetime of fluorescence from the state S1 would be long, and fluorescence from the molecule would be readily controlled by excitation from the state S1 to the state S2 by photo-activation. Therefore, effective use becomes possible for super-resolution. That is, if observation is carried out after dying by such a fluorescence labeler molecule, not only does it become possible to observe a fluorescent image of the sample at high spatial resolution, but it also is possible to selectively dye only particular chemical structures of the biological sample, and it becomes possible to analyze even the detailed chemical structures of the sample.
Moreover, since double-resonance absorption generally only occurs when particular conditions are satisfied, such as polarization state and wavelengths of the two types of light, these conditions can be utilized to learn the structure of the molecule in extremely fine detail. That is, the double-resonance absorption process occurs strongly when there is a strong correlation between polarization direction of the light and orientation direction of the molecule, and when the molecular orientation direction has a particular angle relative to respective polarization directions of the lights of the two wavelengths. Therefore the extent that fluorescence disappears can be varied by irradiating the sample simultaneously with light of two wavelengths and the rotating polarization direction of each respective light. Therefore from such variation, spatial orientation information can be obtained for the tissue under observation. This is also possible by adjustment of the two wavelengths of light.
As mentioned above, according to the technique disclosed in Japanese Patent Application No. Kokai Hei 11-95120, the higher analysis can be realized, in addition to the super high resolution. Moreover, by controlling the timing of period of the irradiation of the two type of lights of wavelength λ1 and wavelength λ2, the S/N ratio can be improved and the restriction of fluorescence can be realized effectively, and thus, the super resolution can be realized more effectively.
A first concrete embodiment of such a super resolution microscope is disclosed in Japanese Patent Application Kokai 2001-100102. In the embodiment, a light (particularly laser light) of wavelength λ1 to excite a fluorescent labeler molecule to the state S1 from the state S0 is employed as a pumping light, and a light of wavelength λ2 to excite the fluorescent labeler molecule to the state S2 from the state S1 is employed as an erasing light. Then, as shown in FIG. 3, the pumping light is irradiated from an optical source 11 and the erasing light is irradiated from an optical source 12. The pumping light is reflected at a dichroic mirror 13, and focused on a sample 15 with a ring optical system 14. Then, the erasing light is turned into a hollow beam with a phase plate 16, and passed through the dichroic mirror 13 and combined with the pumping light spatially. Then, the combined light is focused on the sample 15.
In this case, since fluorescent lights around the optical axis where the intensity of the erasing light is made zero does not almost occur, only fluorescent labeler molecules which belongs to a given narrower region (Δ<0.61 ·λ1/NA, NA: the aperture of the ring optical system 14) than the spread of the pumping light can be observed, so that the super resolution of the microscope can be developed. The phase plate 16 is constructed so that it can shift the phase of the erasing light by point symmetry for the optical axis.
FIG. 5 is a structural view showing another embodiment (second embodiment) of a conventional super resolution microscope. In this embodiment, a laser light emitted from a Nd:YAG laser 21 is split by a half mirror 22. One split beam is introduced to a dichroic mirror 24 via a triple-wave generator 23. The other split beam is introduced to the dichroic mirror 24 via a mirror 25, a Raman shifter 56, a mirror 27 and a phase plate 28, to be combined with the one beam passing through the triple-wave generator 23. The combined light is focused on a sample 35 held with a cover glass 34 on a mobile stage 33 via a condenser lens 29, a pinhole 30, dichroic mirror 31 and an objective lens 32. The phase plate 28 is constructed so that it can shift the phase of the light by point symmetry for the optical axis, as shown in FIG. 6, and the sample 35 is dye with given fluorescent labeler molecules in advance.
A fluorescent light emitted from sample 35 is passed through objective lens 32, and is split from the approach route by a dichroic mirror 31, introduced into a photo multiplier 40 via a pinhole 36, a sharp cut filter 37, a band-pass filter 38, and a notch filter 39. Here, the sharp cut filter 37, the band-pass filter 33, and the notch filter 39 are accommodated in a light-shielded box 41, and the pinhole 36 is formed at the light-shielded box 41.
In the super resolution microscope as shown in FIG. 5, a light (laser light) through the triple-wave generator 23 is employed as a pumping light to excite a fluorescent labeler molecule to the state S1 from the state S0, and a light (laser light) through the Ramen shifter 26 is employed as an erasing light to excite the fluorescent labeler molecule to the state S2 from the state S1. Then, the erasing light is turned into a hollow beam by the phase plate 28, and combined spatially with the pumping light by the dichroic mirror 24. In this case, fluorescent light around the optical axis of the erasing light where the intensity of the erasing light is made zero does not almost occur, and thus, only fluorescent labeler molecules which belong to a narrower region than the spread of the pumping light is observed in high resolution.
Moreover, such a high performance microscope is proposed in Japanese Patent Application No. Kokai Hei 8-184552, where by employing double resonance absorption process through the irradiation for a sample to be observed using plural light beams having their respective wavelengths, the chemical analysis can be realized, in addition to the control of the image to be obtained of the sample.
However, according to various types of investigations by the inventors of the present invention, it was found that the observing principle of the conventional super resolution microscope as shown in the first embodiment is excellent in the spatial resolution, but has some problems in informations to be obtained, that is, the structure-resolving (analysis). That is, in the super resolution microscope, although the pumping light and the erasing light are focused on a sample dyed by fluorescent indicator molecules and then, a fluorescent light from the fluorescent indicator molecule is detected, in this case, the luminance mechanism is based on the following fact. That is, due to irradiating pumping light, the fluorescent molecule with the ground state is excited to a vibration state belonging a high electron state. Thereafter, the excited fluorescent molecule is thermally relaxed to the bottom vibration state of the first electron excited state, and then, de-excited to the ground state through the emission of fluorescence (Kasha rule). In this case, only the information of “where being dyed by the fluorescent indicator molecule within the sample” can be obtained.
Whereas, in a bio-science field, since a biogenic sample is employed, the high resolution and real-time observation for the biogenic activity of the tissue of the sample is strongly desired, and also, such a detail information as about the combining portion and the combining condition of a fluorescent indicator molecule for the tissue is required. Moreover, if possible, without the dying for a sample, the high resolution observation for the biogenic activity of the tissue of the sample is acutely required.
Generally, the reason of dying a biochemical sample with a fluorescent indicator molecule is that in almost biogenic molecules, large energies are required to transit the molecules to their first electron excited states from their ground states and thus, a far ultraviolet beam source is required, but as of now, a far ultraviolet laser source for commercial use is not realized, so that a laser microscope system can not constructed in the far ultraviolet region. Although an xenon lamp or a mercury lamp is employed as a far ultraviolet beam source, the luminance of such a light source is weak and is not practicable because the performance of the light source is inferior to that of a laser source in light divergence and optical polarization.
Also, in the super resolution microscope as shown in the second embodiment, the erasing light which has a larger intensity than the pumping light is focused on the sample dyed by the fluorescent indicator molecules simultaneously or in more or less time interval, and the weak signal is selected and amplified at the photo multiplier while the erasing light being irradiated. In the super resolution microscope as shown in FIG. 5, by disposing the various optical filters etc., and controlling the electrical gate at the detection of the fluorescent signal, the optical scattered components of the erasing light and the pumping light are eliminated.
However, if the erasing light having a large intensity is irradiated onto the sample, side-fluorescence due to the complex multi-photon absorption process can not be neglected. Generally, since the wavelength of the erasing light is set within a longer wavelength region than the absorption region to excite the fluorescent molecule to the first electron excited state from the ground state, principally, no fluorescence is observed. Moreover, since the intensity of the erasing light is controlled so that the two photon absorption process can not be generated and thus, the fluorescence due to the absorption process can not generated, fundamentally, any other photon emission process is observed.
Whereas, if the fluorescent indicator molecules are dispersed in the biogenic sample and combined with given chemical groups, the electron structure of the fluorescent molecules may be changed. As a result, the central wavelength of the absorption region to excite the fluorescent molecules may be shifted in a longer wavelength region, and thus, the fluorescent molecules may be excited by the erasing light, so that weak fluorescence may be generated. Moreover, since the fluorescent indicator molecules are chemically bonded with the biogenic organism of the sample, the electron structure of the biogenic sample may be changed, and thus, a given fluorescence may be generated due to the irradiation of the erasing light.
Since such a side-fluorescence signal is superimposed and mixed with the inherent florescence signal to be observed, it is difficult to distinguish from them with an optical filter and electrical gate control as desired. As a result, the intensity of back ground signal is increased and thus, the super resolution and the image quality may be deteriorated.
Therefore, in the case that the high resolution is required, a new technology capable of eliminating the back ground signal is desired essentially because the observation region per one sampling pixel is narrowed and thus, the number of the fluorescent molecules are decreased within the observation region, the inherent fluorescence signal becomes weak.